A Toll gene is known to be necessary for patterning dorsoventral axis while an embryo is developing in Drosophila (Cell 52, 269-279, 1998; Annu. Rev. Cell Dev. Biol. 12, 393-416, 1996), and for an antifungal immune response in an adult body (Cell 86, 973-983, 1996). The Toll is a type-I transmembrane receptor with an extracellular domain including leucine-rich repeats (LRRs) and this intracellular domain has been elucidated that it demonstrates high homology with that of mammalian interleukin-1 receptor (IL-1R) (Nature 351, 355-356, 1991; Annu. Rev. Cell Dev. Biol. 12, 393-416, 1996; J. Leukoc. Biol. 63, 650-657, 1998).
Recently, mammalian homologs of Toll, called Toll-like receptors (TLR) were identified, and heretofore, 10 families such as TLR2, TLR3, TLR4, TLR6, TLR7, TLR8, TLR9, and TLR10 have been reported (Nature 388, 394-397, 1997; Proc. Natl. Acad. Sci. USA 95, 588-593, 1998; Blood 91, 4020-4027, 1998; Gene 231, 59-65, 1999). This TLR family is known that it recruits IL-1R-associated kinase (IRAK) through MyD88 which is an adapter protein as well as aforementioned IL-1R, and then activates downstream mitogen-activated protein (MAP) kinase and NF-κB which is a nuclear factor (J. Exp. Med. 187, 2097-2101, 1998; Mol. Cell 2, 253-258, 1998; Immunity 11, 115-122, 1999). In addition, it is also believed that the role of TLR family in mammals is related to innate immune recognition as a pattern recognition receptor (PRR) that recognizes the consensus structure of bacteria (Cell 91, 295-298, 1997).
One of pathogen-associated molecular patterns (PAMPs) recognized by the aforementioned PRR is a lipopolysaccharide (LPS) which is a principal component of outer-membrane of Gram-negative bacteria (Cell 91, 295-298, 1997). It is known that host cells are stimulated by the LPS to generate various kinds of inflammatory cytokines such as TNF-α, IL-1 and IL-6 and the like on the host cells (Adv. Immunol. 28, 293-450, 1979, Annu. Rev. Immunol. 13, 437-457, 1995), and LPS captured by LPS-binding protein (LBP) is delivered to CD14 on cell surface (Science 249, 1431-1433, 1990; Annu Rev. Immunol. 13, 437-457, 1995). The present inventors generated knockout mice of TLR4 and reported that TLR4 knockout mice are unresponsive to LPS which is a principal component of the aforementioned outer-membrane of Gram-negative bacteria (J. Immunol. 162, 3749-3752, 1999). They generated TLR2 knockout mice and reported that macrophages of themselves lower the reactivity to Gram-positive bacteria cell walls or peptidoglycan which is a component thereof (Immunity, 11, 443-451, 1999), and that biological reaction is induced via TLR2 and MyD88 signaling pathway (J. Immunol. 164, 554-557, 2000).
Further, the present inventors compared/analyzed TLR6 knockout mice, wild-type mice and TLR2 knockout mice to clarify that TLR6 is a receptor protein specifically recognizing mycoplasma-derived lipoproteln/lipopeptide. They also found full length cDNA (GenBank Accession No. AF245704) for TLR9 and clarified that TLR9 is a receptor protein specifically recognizing bacterial DNA including unmetylated CpG sequence. Moreover, two new members of TLR7 and TLR8 are registered to GenBank (Accession No. AF240467 and AF246971).
On the other hand, imiquimod, which is an immune response-modulator that have been found when screening antiherpesvirus activity, shows antivirus activity and antitumor activity in animal model. It is clarified this agent shows antivirus activity and antiproliferative activity by inducing cytokines such as IFN-α, IL-6 and IL-12, similarly to a secondary induction of IFN-γ in various types of cells (J. Leukoc. Biol. 58, 365-372, 1995; J. Interferon Res, 1989; S2115, Antimicrob. Agents Chemother. 38, 2059-2064, 1994; Am. J. Clin. Pathol. 102, 768-774, 1994), and that imiquimod stimulates NF-κB and MAP kinase (J. Immunol. 165, 5552-5557, 2000; Mol. Cell. Biol. 15, 2207-2218, 1995), as well. In addition, imiquimod and its related compounds are known to inhibit the replication of type 2 herpes simplex virus and cytomegalovirus (J. Infect. Dis. 183, 844-849, 2001; Antimicrob. Agents Chemother. 32, 678-83, 1988). In actual treatment, it is reported that imiquimod is effective for treating pudendal wart (condyloma acuminatum) caused by human papilloma virus (Sex. Transm. Infec. 76, 162-8, 2000), and that resiquimod which is a derivative of imiquimod, abbreviated as R-848, is also effective for treating genital herpes (JAMA 285, 2182-2183, 2001). The structural formulae of these imiquimod and R-848 that are imidazoquinoline group compounds are shown as follows:

TLR families are known to be receptors, involved in recognizing the components of pathogens, and it has been clarified by the present inventors that the components derived from various kinds of bacteria and mycoses are recognized by TLR family, as mentioned above. In other words, the present inventors have already generated TLR2-, TLR4-, TLR6-, and TLR9-deficient mice, and revealed that TLR4, TLR2, TLR6, and TLR9 are receptors which respond to lipopolysaccharide (LPS), to pepridoglycan and lipopeptide derived from Gram-positive bacteria, to polypeptide of mycoplasma similarly to TLR2, and to bacterium DNA including non-methylate CpG sequence, respectively. However, it is still unknown which substances the other TLR family member such as TLR3, TLR7, TLR8 or TLR10 and the like recognizes. In addition, proteins, which can recognize immunopotentiating synthetic compounds, have been unknown.
The object of the present invention is to provide a relationship between each member of TLR family to signaling by stimulation with an immunopotentiating synthetic compound in vivo, particularly a non-human animal model unresponsive to synthetic compounds, wherein a gene function that encodes TLR7 recognizing immunopotentiating synthetic compounds lacks on its genomic locus, said compound being useful to elucidate the relationship of each TLR family member toward signaling by stimulation of immunopotentiating synthetic compounds in vivo, particularly the role of TLR7 in vivo, particularly a non-human animal wherein the gene function of TLR7 lacks on its genomic locus, a screening method for substances for inhibiting or a promoting a response to an immunopotentiating synthetic compound by using the above-mentioned non-human animal models, and a screening method for an immunopotentiating synthetic compound.
The present inventors generated MyD88-deficient mice, and already reported that the macrophages derived from MyD88-deficient mice do not respond to any of TLR ligands when producing cytokines. Since it is not possible to confirm the response in cells of MyD88-deficient mice, various kinds of compounds which might activate cells via TLR were examined. Large number of compounds were screened, and through the process of inducing cytokines from mononuclear leukocytes or macrophages, imidazoquinoline group compounds which might induce humoral immunity and cellular immunity, i.e. imidazoquinoline group compounds such as imiquimod, R-848 and the like which act on various cells and which are known as an immunopotentiator inducing generation of inflammatory cytokines such as IFN-α, IL-6 and IL-12 and the like were treated as target compounds. Meanwhile, the primary structure of the protein of TLR7 is quite similar to that of TLR9 which recognizes microbial DNA. However, the lack of reactivity to microbial DNA is not observed in TLR7-deficient mice as in TLR9-deficient mice. Then, TLR7-deficient mice of TLR family were generated, and the substances recognized by TLR7 were screened subsequently having a TLR7 as a target.
Then, the present inventors stimulated TLR7-deficient mice with R-848 and found that induction of generation of inflammatory cytokines from macrophages, induction of proliferation of B cells, and induction of maturation of dendritic cells were not observed at all. Moreover, in macrophages derived from TLR7-deficient mice, the activation of intracellular signals such as NF-κB, JNK, IRAK by stimulation with R-848 was not observed at all. This suggests that TLR7 is involved not only in recognition of components of pathogen but also of a synthetic compound. Considering that the reactivity to R-848 was completely defective also in MyD88-deficient mice, wherein MyD88 is an adapter molecule that plays an essential role to TLR family signaling pathway, it was thought that TLR7 was an essential receptor for recognition of imidazoquinoline group compounds R-848, and imidazoquinoline group compounds R-848 exerted adjuvanticity by signaling pathway via TLR7-MyD88. As imidazoquinoline group compounds R-848 has an intensive antiviral action and immunopotentiating effect for immune cells, and used for treating actually human pudendal wart caused by papilloma virus, it was thought that TLR7-deficient mice could be not only a very useful model mice for elucidating the action mechanism of imidazoquinoline group compounds, but also to be a very useful model mice for clinical application of synthetic compound agents such as a therapeutic agent for viral infectious diseases, with TLR7 as a target. The present invention has been completed based on the above-mentioned knowledge.